Aging is characterized by reduced skeletal muscle mass (sarcopenia) and increased visceral adipose tissue deposition. These changes in body composition result in frailty, insulin resistance, and cardiovascular disease, which are major health problems in the aging Veteran population. Interleukin-15 (IL-15) is a muscle-derived cytokine with favorable effects on muscle mass, fat mass, and insulin sensitivity in young mice. Circulating IL-15 levels decline with age, in ad lib-fed, but not calorie- restricted rodents, suggesting declines in IL-15 activity contribute to age-associated changes in body composition. However, while IL-15 has favorable effects on body composition in young mice, IL-15 overexpression in aging mice results in increased fat mass and reduced muscle mass compared to controls. IL-15 secretion and bioactivity are modulated by differential association of IL-15 with several forms of the IL-15 alpha receptor subunit (IL-15R1), which can appear as a membrane-bound form (mbIL-15R1) or as one of two soluble forms (sIL-15R1). The latter can differentially affect IL-15 secretion and activity in a tissue- specific manner. Association of IL-15 with sIL-15R1 variants can also differentially inhibit signaling by tumor necrosis factor-1 (TNF-1), another cytokine involved in muscle wasting and insulin resistance. In human subjects, several single-nucleotide polymorphisms (SNPs) have been described in the IL-15R1 gene which impact muscularity, adiposity, and insulin sensitivity. In skeletal muscle, expression of the alternatively-spliced mRNA variant of IL-15R1 (spliced sIL-15R1) declines with age, suggesting a decrease in IL-15/spliced sIL-15R1 complexes leads to age-associated declines in IL-15 levels and activity. The molecular biochemistry of IL-15 and IL-15R1 interactions are complex, tissue-specific, and have not been well-studied in skeletal muscle or adipose tissue, nor in aging systems. Therefore, this study will test the overall hypothesis that IL-15 has opposing effects on body composition in young and old mice due to decreased expression of spliced sIL-15R1 and increased association of IL-15 with the molecularly distinct, proteolytically-shed sIL-15R1 form. The proposed study will utilize retroviral vectors to overexpress IL-15 and IL-15R1 variants in mouse primary and immortalized skeletal myogenic cultures, in order to define the roles of these molecules in IL-15 secretion and bioactivity in muscle. The effects of IL-15/sIL-15R1 complex variants in mouse immortalized adipogenic cultures will also be determined. The effects of IL-15/sIL-15R1 complex variants on TNFR1- mediated TNF-1 signaling in skeletal myogenic and adipogenic cultures will be determined as well. In vivo, the innovative and translational technique of plasmid electroporation into skeletal muscle (EPM) will be used to induce systemic expression of IL-15, sIL-15R1 variants, and a putative superagonistic IL-15/sIL15R1 fusion protein, in young and normally-aging mice. The effects of these molecules on body composition and insulin sensitivity in response to high-fat feeding, sarcopenia, and TNFR1-mediated skeletal myonuclear apoptosis will be determined. The Specific Aims of the project are: 1. Determine the role of IL-15R1 variants in IL-15 stability, secretion and bioactivity in skeletal myogenic and adipogenic cultures. 2. Determine the role of IL-15R1 variants on IL-15 secretion from muscle, body composition, and insulin sensitivity in vivo. 3. Determine the bioactivity of an IL-15/sIL-15R1 fusion protein in muscle and adipose tissue in young and aging mice. These studies will provide mechanistic information which could be exploited pharmacologically to modulate body composition and the pathological consequences of sarcopenia and adiposity in the elderly. PUBLIC HEALTH RELEVANCE: Aging is characterized by decreases in muscle weight and strength, and increased deposition of fat. These changes lead to frailty and insulin resistance (a kind of pre-diabetes), which are major health problems in aging Veterans. Interleukin-15 (IL-15) is a protein that can prevent muscle wasting, obesity, and insulin resistance in young mice. Also, IL-15 levels are lower in old mice than in young mice. Therefore, IL-15 has been suggested as a therapy for the decreases in muscle and increases in fat in the elderly. This notion is too simplistic, however, because recent findings indicate IL-15 has the opposite effect in aging mice. Another set of proteins regulate IL-15, and the amounts of these regulating proteins change during aging. This project will study the regulatory proteins in mice, to determine whether some help IL-15 and some prevent IL-15 from working, and will study how levels of these substances can be controlled during aging. This information will be used in the future to design drugs which can help prevent muscle wasting, obesity, and insulin resistance in the elderly.